Subtilisin enzymes usually refer to extracellular serine endopeptidases from related Bacillus species: for example subtilisin Carlsberg from Bacillus licheniformis (Jacobs et al., Nucleic Acids Res 13: 8913–8926, 1985); subtilisin BPN from Bacillus amyloliquifaciens (Wells et al., Nucleic Acids Res 11: 7911–7925, 1983) and alkaline protease PB92 from Bacillus alcalophilus PB92 (Van Der Laan et al., Appl. Environ. Microbiol. 57, 901–909, 1991) etc. Subtilsin enzymes have been studied extensively in last decades because of their usefulness as additives to detergents, esp. to laundry detergents.
There are several advantages about these subtilisins mentioned above. They usually possess high efficiency and little specificity e.g. they can degrade almost all kinds of proteins. They can exhibit activity at high pH (pH 8–12) and in the presence of some surfactants. In addition they are extracellular enzymes secreted by the bacteria into the medium. Thus they can be isolated without breaking the bacterial cells, which makes the purification process easier and less costly.
To be suitable for use in detergents, proteases must exhibit the following properties:    1. They must possess broad substrate specificity;    2. They must have activity and stability at alkaline pH range.    3. They must be stable at high temperature and in the presence of chelating agents, perborates and surfactants.    4. They must be efficacious at low temperatures (20–40° C.).
However, the yield of subtilisin naturally secreted by Bacillus species is usually low and could not meet the requirement of industry. Fortunately, the application of genetic engineering has greatly enhanced its production yield (Jacobs et al., Gene 152: 69–74, 1995; Zaghloul et al., Enzyme Microb Technol 16: 534–537, 1994). Now subtilisins can be industrially produced. In this patent an expression system based on Bacillus subtilis was successfully used to produce subtilisins with high yield in a short period of time.
Enzyme Production by the Phase φ105 Overexpression System
In a previously established φ105 system (Thornewell et al., Gene 133:47–53, 1993), a defective prophage vector, φ105MU331 was derived for high-level protein over-expression expression in B. subtilis (Leung & Errington, Gene 154(1):1–6, 1995). In this derived system, not only efficient inducible (by heat) transcription of the gene is provided, but also, it prevented the lysis of the host cell. Thus the enzyme produced can be collected easily in the culture media without disruption of the cells, which means the purification steps can be greatly diminished. In addition to this, unlike E. coli, Bacilli are GRAS bacteria, the genes encoding their proteins are also GRAS to animals and thus, human.